The present invention relates to the fields of molecular biology and microbiology, and to the use of genetic techniques to introduce a modified pathway for the production of desired compounds. More specifically, this invention describes genetically engineered biocatalysts possessing an enhanced, or new, ability to transform p-cresol or toluene to p-hydroxybenzoate.
p-Hydroxybenzoate (PHBA) is used as a monomer for synthesizing Liquid Crystal Polymers (LCP). LCP""s are used in electronic connectors and in telecommunication and aerospace applications. LCP resistance to sterilizing radiation suits these materials for use in medical devices as well as in chemical, and food packaging applications. Esters of PHBA also are used as backbone modifiers in other condensation polymers (i.e., polyesters), and are also used to make parabens preservatives.
Chemical synthesis of PHBA is known. For example, JP 05009154 teaches a chemical route using the Kolbe-Schmidt process from tar acid and CO2 involving 1) the extraction of tar acid from a tar naphthalene oil by an aqueous potassium hydroxide, 2) adding phenol to the extracted tar acid potassium salt, 3) removing H2O, and 4) reacting the resultant slurry with CO2. Alternative methods of chemical synthesis are known (see, for example, U.S. Pat. No. 5,399,178; U.S. Pat. No. 4,740,614; and U.S. Pat. No. 3,985,797).
However, chemical synthesis of PHBA is problematic and costly due to the high energy needed for synthesis and the extensive purification of product required. An alternate low cost method with simplified purification would represent an advance in the art. Biological production offers one such low cost, simplified solution to this problem.
Microbiological methods of PHBA synthesis are known. For example, JP 06078780 teaches PHBA preparation by culturing benzoic acid in the presence of microorganisms (preferably Aspergillus) that oxidize benzoic acid to PHBA.
An alternate method of biological production is suggested by bacteria that have an enzymatic pathway for the degradation of toluene and other organics where PHBA is produced as an intermediate. The first enzyme in the toluene degradation pathway is toluene monooxygenase (TMO) and the pathway is referred to as the TMO pathway. The steps of the TMO pathway have been described (Whited and Gibson, J. Bacteriol. 173:3010-3020 (1991)) and are illustrated in FIG. 1. Bacteria that possess the toluene degradation pathway are found in the genus Pseudomonas where Pseudomonas putida, Pseudomonas fluorescens, Pseudomonas aeruginosa and Pseudomonas mendocina are the most commonly utilized species. Other examples of aerobic bacteria that are known to degrade toluene are Burkholderia (Johnson et al., Appl. Environ. Microbiol. 63:4047-4052 (1997)), Mycobacterium (Stephen et al., Appl. Environ. Microbiol. 64:1715-1720 (1998)), Sphingomonas (Zylstra et al., J. Ind. Microbiol. Biotechnol. 19:408-414 (1997)) and Rhodococcus (Kosono et al., Appl. Environ. Microbiol. 63:3282-3285 (1997)). In addition, several different species of anaerobic bacteria are known to utilize toluene (Heider et al., Anarobe 3:1-22 (1997)). Toluene degradation pathways have been highly characterized (Romine et al., In Bioremediation of Chlorinated Polycyclic Aromatic Hydrocarbon Compounds; Hinchee, R. E., E d.; Lewis: Boca Raton, Fla. 1994; pp 271-276) and a number of the genes encoding key enzymes have been cloned and sequenced, including the protocatechuate 3,4-dioxygenase genes (Frazee, J. Bacteriol. 175(19):6194-6202 (1993)), the pcaR regulatory gene from Pseudomonas putida, which is required for the complete degradation of p-hydroxybenzoate (Romero-Steiner et al., J. Bacteriol. 176(18):5771-5779 (1994); Dimarco et al., J. Bacteriol. 176(14):4277-4284 (1994)) and the pobA gene encoding the expression of p-hydroxybenzoate hydroxylase (PHBH), the principal enzyme for the conversion of PHBA to protocatechuate (Wong et al., Microbiology (Reading U. K.) 140(10):2775-2786 (1994); Entsch et al., Gene 71(2):279-291 (1988)).
Bacteria that possess the TMO pathway are useful for degrading toluene and trichloroethylene. They are able to use these and other organics as sole carbon sources where they are transformed through PHBA to ring-opening degradation products (U.S. Pat. No. 5,017,495; U.S. Pat. No. 5,079,166; U.S. Pat. No. 4,910,143). By using the chromosomal TMO pathway, in combination with mutations that prevent PHBA degradation in Pseudomonas mendocina KR1, it has been shown that PHBA can be accumulated by oxidation of toluene (PCT/US98/12072).
Recently, various strains of Pseudomonas possessing the toluene degradation pathways have been used to produce muconic acid via manipulation of growth conditions (U.S. Pat. No. 4,657,863; U.S. Pat. No. 4,968,612). Additionally, strains of Enterobacter with the ability to convert p-cresol to PHBA have been isolated from soil (JP 05328981). Further, JP 05336980 and JP 05336979 disclose isolated strains of Pseudomonas putida with the ability to produce PHBA from p-cresol. Additionly, Miller and coworkers (Green Chem. 1(3):143-152 (1999)) have shown the bioconversion of toluene to PHBA via the construction of a recombinant Pseudomonas putida. Their initial catalyst development focused on Pseudomonas mendocina KR1 for production of PHBA from toluene. However, they were unable to obtain significant accumulation of PHBA from toluene using this strain. This result was due to their inability to obtain a sufficient disruption of PobA activity (the enzyme catalyzing m-hydroxylation of PHBA to protocatechuate in the protocatechuate branch of the xcex2-ketoadipate pathway; see FIG. 1).
Although the presence of the TMO pathway in Pseudomonas mendocina KR1 has been documented (Wright and Olsen, Applied Environ. Microbiol. 60(1):235-242 (1994)), the art has not provided a molecular characterization and sequence of the pcu genes encoding the enzymes that transform p-cresol to PHBA in this organism. The art has also not provided bacterial host cells harboring novel recombinant plasmids encoding the enzymes of p-cresol to PHBA oxidation, together with operably-linked native promoter and regulatory sequences and proteins. Such bacterial host strains, if they lack the enzymes to degrade PHBA further, can accumulate PHBA when cultured in the presence of p-cresol.
As an alternative to culturing cells in the presence of p-cresol, the latter compound can be formed from toluene in cells that additionally harbor plasmid-encoded toluene monooxygenase. A bacterial strain harboring plasmid-encoded tmo and pcu operons has not been fully described in the art, particularly a strain that exceeds the production level of PHBA when compared to plasmid-free Pseudomonas mendocina KR1. In addition, expression of the tmo operon using its native toluene-induced promoter localized upstream of a tmoX gene previously has not been known. Therefore, the problem to be solved is the lack of a fully characterized pcu operon and the availability of a bacterial strain harboring plasmid-encoded tmo and pcu operons to use for the bioproduction of PHBA.
The present invention solves the problem of extensively characterizing the pcu operon by providing cloned, sequenced, and expressed genes of the pcu operon from Pseudomonas mendocina KR-1 that can be transformed into and used to produce PHBA from p-cresol in Pseudomonas putida and Agrobacterium rhizogenes strains that do not normally possess this capability. In addition, transformation of the pcu operon into Pseudomonas mendocina KRC16KDpobA51 supplements the endogenous pcu operon leading to an increase in PHBA production. This increase in PHBA production in Pseudomonas mendocina KRC16KDpobA51 transformed with plasmid-encoded pcu is an improvement over PCT/US98/12072.
The present invention provides a method for the production of PHBA comprising: (i) culturing a Pseudomonas, Agrobacterium or related strain transformed with a pcu operon in a medium containing an aromatic organic substrate, at least one suitable fermentable carbon source, and a nitrogen source, wherein the supplied pcu operon comprises genes encoding the TMO toluene degradation pathway enzymes p-cresol methylhydroxylase and p-hydroxy-benzaldehyde dehydrogenase, the transcriptional activator PcuR, wherein the transformed Pseudomonas or Agrobacterium strain does not produce any detectable p-hydroxybenzoate hydroxylase, whereby PHBA accumulates; and (ii) recovering the PHBA.
The present invention also encompasses the combination of the pcu and tmo operons on a single replicon such that expression of tmo is obtained by transcription from a previously undisclosed toluene or p-cresol induced tmoX promoter, and expression of pcu is obtained by transcription using a previously undisclosed sequence encoding a transcriptional activator.
Another preferred embodiment of the present invention includes the recombinant plasmid pMC4 in Pseudomonas putida DOT-T1. This strain synthesized the highest levels of tmo and pcu-encoded enzymes observed and is described herein.
It has also been found that the heterologous todST proteins that control the induction of toluene dioxygenase pathway induce high levels of expression from the tmo pathway genes, and are useful tools to mediate expression of the catabolic tmo genes and PHBA production in any organism that does not possess these genes.